1. Field of the Invention
The invention is related to monoclonal antibodies which interact with platelet cells to inhibit various functions associated with platelet activation. The principal activity demonstrated by the monoclonal antibodies of the present invention is inhibition of the binding of thrombin to the glycoprotein Ib on the surface of the platelet cells.
2. Description of the Related Art
Various articles of the scientific and/or patent literature are cited throughout this document. Each of the articles referred to are hereby incorporated in their entirety by such reference.
The essential functions of platelets involve their response to a variety of stimuli. One of the most important physiologic stimuli is alpha thrombin, a coagulation serine protease, derived from prothrombin by cleavage by coagulation factor Xa. Thrombin plays a key role in blood coagulation and thrombosis (pathological coagulation). Thrombin is a central bioregulatory enzyme in hemostasis. It catalyzes the conversion of fibrinogen to fibrin and is responsible for the activation of coagulation factors V, VIII and XIII. Thrombin may also interact with blood vessel walls causing vasoconstriction and may even mediate leukocyte adherence.
Thrombin is the most potent physiologic stimulus which induces platelet activation. The reaction of thrombin with platelets represents an unusual agonist-receptor interaction. Although stimulation of platelets requires the catalytic activity of thrombin, a major protein, the glycoprotein Ib (GPIb), has been isolated on the platelet surface to which thrombin binds. High affinity thrombin binding to GPIb induces platelet activation; this includes platelet shape change, ADP and serotonin secretion, alpha granule release, lysosomal release, the conversion of arachidonic acid to thromboxane A2 and irreversible platelet aggregation. Thromboxane A2 acts as a potent vasoconstrictor and causes aggregation of platelets.
The role of thrombin in arterial thrombosis and in thrombolytic therapy has been clearly defined. Thrombin causes platelet activation and aggregation which is resistant to conventional thrombolytic therapy. Thus, any agent which could interfere with high affinity thrombin binding to the GPIb on the platelet would be an important tool in treating patients with acute arterial thrombosis and could be efficacious in ameliorating the effects of thrombin in arterial thrombus formation and the resultant resistance of arterial thrombi to thrombolytic therapy.
Monoclonal antibodies have been identified which inhibit platelet functions induced by ristocetin or thrombin. One such antibody, TM-60, inhibits binding of von Willebrand factor to platelets in the presence of ristocetin and inhibited the release of adenosine diphosphate (ADP) by thrombin. The authors show that this antibody immunoprecipitates GPIb. It is important to note that this antibody has very little effect on thrombin induced platelet aggregation as shown in Thrombosis Research, volume 39, page 751-759, 1985. The antibody totally inhibited yon Willebrand factor binding to platelets. No data was shown concerning binding of thrombin to platelets. In a subsequent publication in Thrombosis and Hemostasis, volume 55, pages 162-167, 1986, the authors showed that there was a thrombin binding site on human platelet GPIb. The authors do not disclose that inhibition of binding of thrombin to GPIb is mediated by their antibody and gave no further functional information concerning this antibody. In another publication in Thrombosis and Hemostasis, volume 63, pages 122-126, 1990, the authors showed data concerning the ability of the TM-60 antibody to inhibit platelet aggregation induced by ristocetin and thrombin. A subsequent paper in Blood, volume 77, pages 1740-1748, 1991 showed that this antibody can only inhibit approximately 50% of high affinity thrombin binding to the platelet.
Another monoclonal antibody to human platelet GPIb, designated antibody SZ-2, inhibited both ristocetin- and collagen- induced aggregation of platelets (C. Ruan et al., in Monoclonal Antibodies and Blood Platelets, INSERM Symposium No. 27, pp. 59-68, ed. J. L. McGregor, c. 1986 by Elsiver Science Publishers BV.). A third antibody to GPIb is designated VM 16d and blocks thrombin-induced platelet aggregation at low doses of thrombin, 0.05 u/ml. This antibody recognized a site on the GPIb; however, the authors did not demonstrate that this antibody inhibited high affinity binding of thrombin to platelets nor did they show that their antibody could inhibit thrombin-induced changes in calcium flux or ADP release (C. Ruan et al., Thrombosis Research 62:673-684 (1991)). Another antibody which modifies alpha thrombin function was described by DeMarco (DeMarco et al., J. Biol. Chem. 266:23776-23783 (1991)). This antibody, designated LJ-Ib10, inhibits high affinity thrombin binding to platelets, but does not interfere with the moderate to low affinity binding of thrombin to platelets. Thus, LJ-Ib10 is able to inhibit at most only 50% of the total measurable thrombin binding to platelets. Interference of thrombin binding to platelets by LJ-Ib10 results in decreased fibrinogen binding to platelets and also inhibits the thrombin-mediated calcium flux across the platelet membrane and release of ADP from the cells. The antibody LJ-Ib10 only partially inhibited thrombin-induced platelet aggregation.
The antibody F124H12 (4H12), described in the present application, is similar to the LJ-Ib10 antibody, but demonstrates advantageous properties over that and the other previously described antibodies to GPIb. 4H12 completely inhibits the high affinity binding of thrombin to GPIb, as shown for LJIb-10. As a result, 4H12 completely inhibits the binding of fibrinogen to platelets that results from thrombin activation of the cells. 4H12 also completely inhibits the other physiologic responses of platelets to thrombin. Platelets incubated with 4H12 prior to thrombin exposure retain their unactivated shape, do not exhibit a flux of calcium across the membrane and do not release ADP, serotonin, lysosomes or alpha granules, do not convert arachidonate to thromboxane, and most importantly, the platelets do not aggregate.